ANTIHYPERLIPIDAEMIC AND ANTIOXIDANT ACTIVITIES OF EXTRACTS OF DIFFERENT PARTS OF AVERRHOA CARAMBOLA AND ELUCIDATION OF THEIR MECHANISMS OF ACTION SULTAN AYESH MOHAMMED SAGHIR UNIVERSITY SAINS MALAYSIA 2015
ANTIHYPERLIPIDAEMIC AND ANTIOXIDANT
ACTIVITIES OF EXTRACTS OF DIFFERENT PARTS OF
AVERRHOA CARAMBOLA AND ELUCIDATION OF
THEIR MECHANISMS OF ACTION
SULTAN AYESH MOHAMMED SAGHIR
UNIVERSITY SAINS MALAYSIA
2015
ANTIHYPERLIPIDAEMIC AND ANTIOXIDANT
ACTIVITIES OF EXTRACTS OF DIFFERENT PARTS OF
AVERRHOA CARAMBOLA AND ELUCIDATION OF
THEIR MECHANISMS OF ACTION
By
SULTAN AYESH MOHAMMED SAGHIR
Thesis submitted in fulfillment of the
requirements for the degree of
Doctor of Philosophy
UNIVERSITY SAINS MALAYSIA
OCTOBER 2015
DEDICATION
In the name of ALLAH, The Most Gracious, The
Most Merciful
THIS THESIS IS DEDICATED
TO
MY MOTHER AND FATHER FOR DOING THEIR BEST
TO EDUCATE ME,
MY WIFE EMAN AND MY DAUGHTERS MANAL, HUDA, DUA`A AND
ALAA FOR THEIR PATIENCE, UNDERSTANDING, LOVE, AND
SINCERITY
ii
ACKNOWLEDGMENT
All praises to the Almighty Allah, Who is omnipotent and all giving, for affording me
the strength and determination to complete this study. I would like to express my deepest
gratitude and sincere thanks to my supervisor Dr. Vikneswaran Murugaiyah for his
guidance, valuable suggestions, continued support and encouragement throughout this
work as well as the writing of this thesis. I am particularly grateful my co-supervisor
Proffesor Dr. Amirin Sadikun, who provided me the needed support, good comments
and valuable suggestions. I wish to express my thanks to the Ministry of Public Health
and Population, Yemen for their help and giving me a chance to complete my study and
I would like to thank Universiti Sains Malaysia, Malaysia for their support (Graduate
assistant) during my study and providing all the facilities required to do this work. I take
this opportunity to thank Associate Prof. Dr. Gurjeet Kaur, Pathologist, INFORM
Universiti Sains Malaysia, for her valuable help in interpreting the histopathology
results. Also, I wish to thank Animal house unit staff, main campus, University Sains
Malaysia and Mr. Rusli Hassan who manages the transit room in School
of Pharmaceutical Sciences for their valuable assistance in animal studies. I would like
to acknowledge the following individuals: Manimegalai, Majed Kacem Al-mansoub,
Vageesh Revadigar, Jayadhisan Muniandy, Pravin Kumar, Khaw Kooi Yeong,
Mohammad Razak Hamdan, Ahmed Anuar, Abdul Hakim Memon, Mohammed Shahrul
Ridzwan, Christapher Varghese, Mohammed Ayesh, Fouad Ayesh, Motaher Ayesh,
Rhadhya Sahal, Fisal Jamaludin, Selvamani Nair, Fouad Saleh Al-Suede and
Mohammed Ali Ahmed Saeed, as well as Dr. Mahfoud Abdulghani Al-Musali and
iii
Dr. Omar Saeed Al-Salahi for their contribution and support. I wish to express my
thanks to my mother and father who always pray for me, for my wife who supports and
encourages me, for my kids who make me laugh and happy. Finally, I wish to
acknowledge all those who have cooperated with me during this endeavor, in all lab
work and who have read, reviewed and offered numerous helpful suggestions and
proposed corrections.
iv
TABLE OF CONTENTS
Content………………….. ................................................................................. …… Page
TITLE……………………………………………………………….……………………...i
ACKNOWLEDGMENT ...................................................................................................... ii
TABLE OF CONTENTS ................................................................................................. ...iv
LIST OF TABLES ............................................................................................................. xv
LIST OF FIGURES ........................................................................................................... xix
LIST OF SYMBOLS ........................................................................................................ xxx
LIST OF ABBREVIATIONS ......................................................................................... xxxi
ABSTRAK ..................................................................................................................... xxxv
ABSTRACT ................................................................................................................. xxxvii
CHAPTER 1: INTRODUCTION ..................................................................................... 1
1.1 Background ................................................................................................................... 1
1.2 Therapeutic challenges .................................................................................................. 3
1.3 Problem statements ........................................................................................................ 4
1.4 Objectives ...................................................................................................................... 4
1.5 Flow chart of the study .................................................................................................. 6
CHAPTER 2: LITERATURE REVIEW ......................................................................... 7
2.1 Lipids ............................................................................................................................ 7
2.1.1 Fatty acids ........................................................................................................... 7
2.1.2 Phospholipids ...................................................................................................... 8
2.1.3 Triglycerides ....................................................................................................... 8
v
2.1.4 Cholesterol and cholesterol esters ....................................................................... 9
2.2 Lipoproteins ................................................................................................................ 10
2.3 Bile acids .................................................................................................................... 12
2.4 Cholesterol biosynthesis ............................................................................................. 15
2.5 Digestion and absorption of lipids .............................................................................. 17
2.5.1 Digestion and absorption of cholesterol ............................................................ 17
2.5.1.1 Cholesterol and bile acid cross-talk ......................................................... 18
2.5.2 Cholesterol excretion ........................................................................................ 19
2.5.3 Digestion and absorption of triglycerides ......................................................... 19
2.5.4 Digestion and absorption of phospholipids ....................................................... 20
2.6 Lipid metabolic pathways ........................................................................................... 20
2.6.1 Exogenous pathway .......................................................................................... 21
2.6.2 Endogenous pathway ........................................................................................ 22
2.6.3 Reverse cholesterol transport pathway ............................................................. 24
2.7 Hyperlipidaemia: classification and causes ................................................................ 26
2.8 Common approaches used to study hyperlipidaemia ................................................. 27
2.8.1 Chemicals-induced acute hyperlipidaemic model ............................................ 27
2.8.2 High fat diet-induced chronic hyperlipidaemic model...................................... 28
2.9 Lipids lowering agents................................................................................................ 29
2.10 Natural products as source of antihyperlipidaemic agents ....................................... 31
2.10.1 General consideration ..................................................................................... 31
2.10.2 Medicinal plants in hyperlipidaemia .............................................................. 32
2.11 In vitro and in vivo antioxidants................................................................................ 33
vi
2.12 Toxicity study ........................................................................................................... 35
2.13 Averrhoa carambola ................................................................................................. 37
2.13.1 Taxonomy of Averrhoa carambola ................................................................. 37
2.13.2 Plant description ............................................................................................. 37
2.13.3 Traditional uses of Averrhoa carambola ........................................................ 39
2.13.4 Pharmacological and toxicological aspects of Averrhoa carambola ............. 40
2.13.4.1 Antioxidant capacity ............................................................................. 40
2.13.4.2 Anti-inflammatory activity .................................................................... 41
2.13.4.3 Acetylcholinesterase inhibitory activity ................................................ 42
2.13.4.4 Antimicrobial and antifungal activity ................................................... 42
2.13.4.5 Cytotoxicity activity .............................................................................. 43
2.13.4.6 Anti-ulcer activity ................................................................................. 43
2.13.4.7 Negative inotropic and chronotropic effect ........................................... 43
2.13.4.8 Electrophysiological effects .................................................................. 44
2.13.4.9 Hypotensive activity .............................................................................. 45
2.13.4.10 Hypocholesterolemic activity ............................................................. 45
2.13.4.11 Hypoglycaemic activity ....................................................................... 46
2.13.4.12 Nephrotoxic effect .............................................................................. 48
2.13.4.13 Neurotoxic effect ................................................................................ 48
2.13.5 Phytochemistry ............................................................................................... 49
2.13.6 Clinical studies ................................................................................................ 56
vii
CHAPTER 3: MATERIALS AND METHODS ............................................................ 58
3.1 Materials and equipments ............................................................................................ 58
3.2 Collection and preparation of plant materials............................................................. 61
3.2.1 Fractionation of methanolic extract of Averrhoa carambola leaf by liquid-
liquid partition .................................................................................................... 62
3.3 Evaluation of antihyperlipidaemic effect ................................................................... 64
3.3.1 Evaluation of antihyperlipidaemic effect of methanol and aqueous extracts of
different parts of Averrhoa carambola plants in poloxamer 407-induced acute
hyperlipidaemic rat model ................................................................................. 64
3.3.1.1 Animals .................................................................................................... 64
3.3.1.2 Induction of hyperlipidaemia ................................................................... 64
3.3.1.3 Experimental design ................................................................................ 65
3.3.1.4 Analysis of lipid profile ........................................................................... 66
3.3.2 Evaluation of antihyperlipidaemic effect of different doses of
methanolic extract of Averrhoa carambola leaf in high fat diet-induced chronic
hyperlipidaemic rats model and subsequently a dose-response study ............... 67
3.3.2.1 Induction of hyperlipidaemia in rats ........................................................ 67
3.3.2.2 Experimental design ................................................................................ 68
3.3.2.3 Analysis of lipid profile ........................................................................... 69
3.3.2.4 Body weight and relative liver weight ..................................................... 70
3.3.2.5 Body mass index ...................................................................................... 70
3.3.2.6 Daily food intake ..................................................................................... 70
3.3.2.7 Faecal dry weight ..................................................................................... 71
viii
3.3.2.8 Relative organ weight .............................................................................. 71
3.3.2.9 Histopathology of liver tissue .................................................................. 71
3.3.3 Evaluation of antihyperlipidaemic effect of different fractions of
methanolic extract of Averrhoa carambola leaf using poloxamer-407 induced
acute hyperlipidaemic rats model ...................................................................... 72
3.3.3.1 Induction of hyperlipidaemia in rats ............................................................... 72
3.3.3.2 Experimental design ....................................................................................... 72
3.4 Evaluation of antioxidant activity............................................................................... 73
3.4.1 Evaluation of antioxidant activity of methanolic and aqueous extracts of
different parts of Averrhoa carambola using different in vitro assays .............. 73
3.4.1.1 Determination of total phenolic content .................................................. 73
3.4.1.2 Determination of total flavonoid content ................................................. 74
3.4.1.3 Ferric reducing antioxidant power assay ................................................. 74
3.4.1.4 DPPH free radical scavenging assay ....................................................... 75
3.4.1.5 ABTS radical scavenging assay .............................................................. 76
3.4.2 Evaluation of antioxidant activity of different fractions of methanolic extract of
Averrhoa carambola leaf using different in vitro assays ................................... 76
3.4.2.1 Pearson correlations coefficient analysis ................................................. 77
3.5 Mechanistic study of antihyperlipidaemic effect of methanolic extract of Averrhoa
carambola leaf and its bioactive ethyl acetate fraction ............................................... 77
3.5.1 Assessment of inhibitory activity of methanolic extract of Averrhoa carambola
leaf on HMG-CoA reductase and pancreatic lipase enzymes ............................ 77
3.5.1.1 Effect on HMG-CoA reductase activity .................................................. 77
ix
3.5.1.2 Effect on pancreatic lipase activity ......................................................... 79
3.5.2 Assessment of inhibitory activity of ethyl acetate fraction of methanolic extract
of Averrhoa carambola leaf on HMG-CoA reductase and pancreatic lipase
enzymes.............................................................................................................. 79
3.5.3 Assessment of in vivo antioxidant activity in liver homogenate and serum
samples of rats treated with methanolic extract of Averrhoa carambola leaf
………………………………………………………………………………..80
3.5.3.1 Preparation of liver homogenate and serum samples .............................. 80
3.5.3.2 Determination of total protein of liver homogenates and serum samples81
3.5.3.3 Evaluation of lipid peroxidation using thiobarbituric acid reactive
substances (TBARS) assay .................................................................... 81
3.5.3.4 Superoxide dismutase assay .................................................................... 82
3.5.3.5 Reduced glutathione assay ....................................................................... 83
3.5.3.6 Glutathione peroxidase assay .................................................................. 84
3.5.3.7 Catalase assay .......................................................................................... 85
3.5.4 Evaluation of effect of Averrhoa carambola leaf methanolic extract on liver
total cholesterol and triglycerides ...................................................................... 86
3.5.4.1 Extraction of lipids from liver samples ................................................... 86
3.5.4.2 Determination of cholesterol and triglycerides in liver tissues ............... 87
3.5.5 Evaluation of effect of Averrhoa carambola leaf methanolic extract on lipids
and bile acids excretions .................................................................................... 87
3.5.5.1 Extraction of lipids in faeces samples ..................................................... 87
3.5.5.2 Determination of cholesterol in faeces samples ...................................... 88
x
3.5.5.3 Extraction of faecal bile acids ................................................................. 88
3.5.5.4 Determination of faecal bile acid ............................................................. 88
3.6 Toxicological evaluation of extracts of Averrhoa carambola ..................................... 89
3.6.1 Assessment of in vitro cytotoxicity of methanolic and aqueous extracts of
different parts of Averrhoa carambola .............................................................. 89
3.6.1.1 Cell lines and cell culture maintenance ................................................... 89
3.6.1.2 Cell viability assay ................................................................................... 89
3.6.2 Evaluation of acute and sub-chronic toxicity of methanolic extract of Averrhoa
carambola leaf ................................................................................................... 90
3.6.2.1 Experimental animals .............................................................................. 90
3.6.2.2 Evaluation of acute toxicity of methanolic extract of Averrhoa carambola
leaf .......................................................................................................... 91
3.6.3 Evaluation of sub-chronic toxicity of methanolic extract of Averrhao
carambola leaf ................................................................................................... 92
3.6.3.1 Histopathological assessment of liver and kidney tissue samples ........... 92
3.6.4 Statistical analysis ............................................................................................. 93
3.7 Standardization and quantification of selected biomarker in methanolic extract of
Averrhoa carambola leaf and its ethyl acetate fraction using HPLC method ............. 93
3.7.1 Development and validation of HPLC method ................................................. 93
3.7.1.1 Preparation of samples and standards for HPLC analysis ....................... 93
3.7.1.2 Chromatographic conditions .................................................................... 94
3.7.1.3 Linearity, limit of detection (LOD) and limit of quantification (LOQ) .. 95
3.7.1.4 Within-day, between-day accuracy and precision ................................... 95
xi
3.7.1.5 Recovery .................................................................................................. 96
3.7.2 Analysis and standardization of selected biomarker in methanolic extract of
Averrhoa carambola leaf and its ethyl acetate fraction ..................................... 97
CHAPTER 4: RESULTS ................................................................................................. 98
4.1 Extraction and fractionation yields .............................................................................. 98
4.2 Antihyperlipidaemic effect of Averrhoa carambola ................................................... 99
4.2.1 Antihyperlipidaemic effect of methanolic and aqueous extracts of different
parts of Averrhoa carambola in poloxamer-407 induced acute hyperlipidaemic
rats ...................................................................................................................... 99
4.2.2 Antihyperlipidaemic effect of methanolic extract of Averrhoa carambola leaf
in high fat diet-induced chronic hyperlipidaemic rats and subsequently a dose
response study .................................................................................................. 115
4.2.2.1 Antihyperlipidaemic effect of methanolic extract of Averrhoa carambola
leaf in normal rat .................................................................................. 115
4.2.2.2 Antihyperlipidaemic effect of methanolic extract of Averrhoa carambola
leaf in high fat diet-induced chronic hyperlipidaemic rat .................... 122
4.2.2.3 Effects of methanolic extract of Averrhoa carambola leaf on rat’s body
weight, body mass index, food intake, faecal dry weight, and relative
organ weight of normal rats ................................................................. 130
4.2.2.4 Effects of methanolic extract of Averrhoa carambola leaf on rat’s body
weight, body mass index, food intake, faecal dry weight, and relative
organ weight of high fat diet-induced chronic hyperlipidaemic rats…136
4.2.2.5 Histopathological analysis of liver tissues ............................................ 143
xii
4.2.3 Antihyperlipidaemic effect of different fractions of methanolic extract of
Averrhoa carambola leaf in poloxamer-407 induced acute hyperlipidaemic
rats……………………………………………………………………………146
4.3 Antioxidant activities ................................................................................................ 153
4.3.1 Antioxidant activity of methanolic and aqueous extracts of different parts of
Averrhoa carambola ........................................................................................ 153
4.3.1.1 Analysis of relationship between TPC and TFC against antioxidant and
antihyperlipidaemic activities of methanolic and aqueous extracts of
different parts of Averrhoa carambola ................................................ 156
4.3.2 Antioxidant activity of different fractions of methanolic extract of Averrhoa
carambola leaf ................................................................................................. 157
4.3.2.1 Analysis of relationship between TPC and TFC against antioxidant and
antihyperlipidaemic activities of different fractions of methanolic extract
of Averrhoa carambola leaf ................................................................. 159
4.4 Mechanism of antihyperlipidaemic effect of methanolic extract of Averrhoa
carambola leaf and its ethyl acetate fraction ............................................................. 160
4.4.1 Inhibitory activity of methanolic extract of Averrhoa carambola leaf on HMG-
CoA reductase and pancreatic lipase ............................................................... 160
4.4.2 Inhibitory activity of ethyl acetate fraction on HMG-CoA reductase and
pancreatic lipase enzymes ................................................................................ 162
4.4.3 In vivo antioxidant activity of methanolic extract of Averrhoa carambola
leaf……………………………………………………………………………163
xiii
4.4.3.1 In vivo antioxidant effect of methanolic extract of Averrhoa carambola
leaf in normal rat model ....................................................................... 163
4.4.3.2 In vivo antioxidant activity of methanolic extract of Averrhoa carambola
leaf in high fat diet-induced chronic hyperlipidaemic rats model........ 165
4.4.4 Effect of methanolic extract of Averrhoa carambola leaf on liver total
cholesterol and triglycerides ............................................................................ 169
4.4.4.1 Effect of methanolic extract of Averrhoa carambola leaf on liver total
cholesterol and triglycerides of normal rats ......................................... 169
4.4.4.2 Effect of methanolic extract of Averrhoa carambola leaf on liver total
cholesterol and triglycerides levels of high fat diet-induced
hyperlipidaemic rats ............................................................................. 170
4.4.5 Effect of methanolic extract of Averrhoa carambola leaf on faecal lipids and
bile acids .......................................................................................................... 172
4.4.5.1 Effect of methanolic extract of Averrhoa carambola leaf on faecal lipids
and bile acids excretions of normal rats ............................................... 172
4.4.5.2 Effect of methanolic extract of Averrhoa carambola leaf on faecal lipids
and bile acids excretions of high fat diet-induced chronic
hyperlipidaemic rats ............................................................................. 177
4.5 Toxicological evaluation ........................................................................................... 183
4.5.1 Cytotoxicity of methanolic and aqueous extracts of different parts of Averrhoa
carambola ........................................................................................................ 183
4.5.2 Acute toxicity of methanolic extract of Averrhoa carambola leaf ................. 185
4.5.3 Sub-chronic toxicity of methanolic extract of Averrhoa carambola leaf ....... 186
xiv
4.5.3.1 Histopathological study of liver and kidney tissue samples .................. 195
4.6 Standardization and quantification of apigenin in methanolic extract of Averrhoa
carambola leaf and its ethyl acetate fraction ............................................................. 200
4.6.1 HPLC method validation ................................................................................ 200
4.6.2 HPLC-UV analysis of methanolic extract of Averrhoa carambola leaf and its
ethyl acetate fraction ........................................................................................ 204
CHAPTER 5: DISCUSSION ......................................................................................... 207
CHAPTER 6: CONCLUSION ...................................................................................... 234
6.1 Conclusion…...……………………………………………………………………..234
6.2 Limitation .................................................................................................................. 236
6.3 Future work ............................................................................................................... 236
REFERENCES ............................................................................................................... 237
APPENDICES................................................................................................................. 263
APPENDIX A.................................................................................................................. 263
APPENDIX B .................................................................................................................. 267
APPENDIX C.................................................................................................................. 274
xv
LIST OF TABLES
Table No. Content Page
Table 2.1 Physical properties of plasma lipoproteins………………………................ 12
Table 2.2 Mechanisms of action and side effects of common lipid lowering
drugs………………………………………………………………………..
30
Table 2.3 Traditional uses of different parts of Averrhoa carambola plant………….. 39
Table 2.4 Chemical constituents isolated from Averrhoa carambola…………….….….. 49
Table 3.1 List of chemicals…………………………………………………….….….... 58
Table 3.2 List of reagents and kits…………………………...……………………........ 59
Table 3.3 List of drugs………………………………………..…………..……………. 59
Table 3.4 List of instruments………………………………………………….............. 60
Table 3.5 Preparation of reaction mixtures for measuring HMG-CoA reductase
activity………………………………………………….……………............
78
Table 3.6 Reaction mixtures for the measurement of SOD………..……….………..... 82
Table 3.7 Preparation of samples for measurement of GSH………………..…….….... 83
Table 3.8 HPLC gradient program for apigenin analysis……….…………………….. 94
Table 4.1 Percentage of yields of different fractions of methanolic extract of
Averrhoa carambola leaf………….……………………………...................
98
Table 4.2 The effect of long-term administration of various doses of methanolic
extracts of Averrhoa carambola leaf on relative organ weight of normal
rats after 5 weeks treatment............................................................................
135
xvi
Table 4.3 The effect of long-term administration of various doses of methanolic
extracts of Averrhoa carambola leaf on relative organ weight of high fat
diet-induced chronic hyperlipidaemic rats…………….……………….........
142
Table 4.4 Antioxidant activities of methanolic and aqueous extracts of different parts
of Averrhoa carambola…………………………………………..…....................
155
Table 4.5 Correlation coefficients between TPC and TFC against antioxidant activity
and lipid parameters of methanolic and aqueous extracts of different parts
of Averrhoa carambola……………………………………………………..…......
157
Table 4.6 Antioxidant activity of different fractions of methanolic extract of
Averrhoa carambola leaf……………………………………………………
158
Table 4.7 Correlation coefficients between TPC and TFC against antioxidant activity
and lipid parameters of different fractions of methanolic extract of
Averrhoa carambola leaf……………………………………………………
159
Table 4.8 Effect of long term intake of methanolic extracts of Averrhoa carambola
leaf on selected in vivo antioxidant parameters in liver of normal
rats…………………………………..……………………………….………
164
Table 4.9 Effect of long term intake of methanolic extract of Averrhoa carambola
leaf on selected in vivo antioxidant parameters in serum of normal
rats……………………………………………………….............................
164
Table 4.10 Effect of long term intake of different doses of methanolic extracts of
Averrhoa carambola leaf on selected in vivo antioxidant parameters in
liver of high fat diet-induced chronic hyperlipidaemic rats ……………….
167
xvii
Table 4.11 Effect of long term intake of different doses of methanolic extracts of
Averrhoa carambola leaf on selected in vivo antioxidant parameters in
serum of high fat diet-induced chronic hyperlipidaemic
rats….............................................................................................................
168
Table 4.12 The effect of methanol and aqueous extracts of different parts of Averrhoa
carambola on cell viability of various cell lines at 50 and 100 µg/mL in
comparison with the standards……………………………………….…….
184
Table 4.13 Relative organ weight of female rats treated with different doses of
methanolic extract of Averrhoa carambola leaf for 28
days………….................................................................................................
188
Table 4.14 Relative organ weight of male rats treated with different doses of
methanolic extract of Averrhoa carambola leaf for 28
days………………...... …………………………………………………….
189
Table 4.15 Haematological parameters for female rats treated with different
doses of methanolic extract of Averrhoa carambola leaf for 28
days………………………………………………………………….……....
191
Table 4.16 Haematological parameters for male rats treated with different doses of
methanolic extract of Averrhoa carambola leaf for 28 days…………..........
192
Table 4.17 Biochemical parameters for female rats treated with different doses of
methanolic extract of Averrhoa carambola leaf for 28 days……..…………
193
Table 4.18 Biochemical parameters for male rats treated with different doses of
methanolic extract of Averrhoa carambola leaf for 28 days……..…………
194
xviii
Table 4.19 LOD, LOQ and linearity of standard curves for apigenin…………………. 200
Table 4.20 Recovery precision and accuracy values for apigenin……………….……... 203
Table 4.21 Within-day and between-day precision and accuracy value for
apigenin……………………………………………………….......................
203
Table 4.22 Contents of apigenin in methanolic extract of Averrhoa carambola leaf and
its ethyl acetate fraction……………………………………………..............
204
xix
LIST OF FIGURES
Figure No. Content Page
Figure 1.1 Flow chart of the study…………………………………………….….….. 6
Figure 2.1 Simple outlines of the classic and alternative pathways in bile acids
synthesis………………………………………………………………….
14
Figure 2.2 Biosynthesis of cholesterol, triglycerides and phospholipids…………..… 16
Figure 2.3 Exogenous pathway of lipid metabolism…………………………..…....... 22
Figure 2.4 Endogenous pathway of lipid metabolism………………………………... 24
Figure 2.5 Reverse cholesterol transport pathway……………………………..…….. 26
Figure 2.6 Averrhoa carambola tree…………………..……………………………... 38
Figure 2.7 Averrhoa carambola plant parts used in this study………….…….……... 38
Figure 3.1: Flow chart of fractionation of Averrhoa carambola leaf methanolic
extract………………………………………...…………………………...
63
Figure 4.1 Effect of methanolic extract of different parts of Averrhoa carambola on
total cholesterol level of p-407 induced acute hyperlipidaemic
rats………………………………………………….……………………..
101
Figure 4.2 Figure 4.2: Percentage changes of total cholesterol level of p-407 induced acute
hyperlipidaemic rats after treated with different parts of methanolic
extracts of Averrhoa carambola………………………………….…………….
102
Figure 4.3 Effect of methanolic extract of different parts of Averrhoa carambola on
triglycerides level of p-407 induced acute hyperlipidaemic
rats……………………………………………………..…………………..
103
xx
Figure 4.4 Percentage changes of triglycerides level of p-407 induced
hyperlipidaemic rats after treated with different parts of methanolic
extracts of Averrhoa carambola……………………………………………….
104
Figure 4.5 LDL-C level of p-407 induced hyperlipidaemic rats after treatment with
different parts of methanolic extracts of Averrhoa carambola….………...
105
Figure 4.6 HDL-C level in p-407 induced hyperlipidaemic rats after treated with
different parts of methanolic extracts of Averrhoa carambola….…...……
106
Figure 4.7 VLDL-C level of p-407 induced hyperlipidaemic rats after treated with
different parts of methanolic extracts of Averrhoa carambola……………
107
Figure 4.8 AI level of p-407 induced hyperlipidaemic rats after treated with
different parts of methanolic extracts of Averrhoa carambola……………
107
Figure 4.9 Effect of aqueous extract of different parts of Averrhoa carambola on
total cholesterol level of p-407 induced acute hyperlipidaemic rats…..…
109
Figure 4.10 Percentage changes of total cholesterol level of p-407 induced acute
hyperlipidaemic rats after treated with different parts of aqueous
extracts of Averrhoa carambola……….……………………………………...
110
Figure 4.11 Effect of aqueous extract of different parts of Averrhoa carambola on
triglycerides level of p-407 induced acute hyperlipidaemic
rats………………………………………………………………………..
111
Figure 4.12 Percentage changes of triglycerides level of p-407 induced acute
hyperlipidaemic rats after treated with different parts of Averrhoa
carambola aqueous extracts……………………………………………...
112
xxi
Figure 4.13 LDL-C level of p-407 induced acute hyperlipidaemic rats after treated
with different parts of aqueous extracts of Averrhoa
carambola……………………………………………………………..…………
113
Figure 4.14 HDL-C level of p-407 induced acute hyperlipidaemic rats after treated
with different parts of aqueous extracts of Averrhoa
carambola……………………………………………………………………..…
113
Figure 4.15 VLDL-C level of p-407 induced acute hyperlipidaemic rats after treated
with different parts of aqueous extracts of Averrhoa
carambola………………………………………………………………………..
114
Figure 4.16 AI level of p-407 induced acute hyperlipidaemic rats after treatmant
with different parts of aqueous extracts of Averrhoa carambola…………
114
Figure 4.17 Effect of 1000 mg/kg of methanolic extract of Averrhoa carambola leaf
on total cholesterol level of normal rats after 5 weeks
treatment…………………………………………………..……..……....
116
Figure 4.18 Percentage changes of total cholesterol level of normal rats after
treatment with 1000 mg/kg of methanolic extract of Averrhoa
carambola leaf……………………………………..…...……….……….
117
Figure 4.19 Effect of 1000 mg/kg of methanolic extract of Averrhoa carambola leaf
on triglycerides level of normal rats after 5 weeks treatment…....…..…..
118
Figure 4.20 Percentage changes of triglycerides level of normal rats after treatment
with 1000 mg/kg methanolic extract of Averrhoa carambola leaf for 5
weeks………………………………………………………………..……
119
xxii
Figure 4.21 LDL-C level of normal rats after treatment with 1000 mg/kg methanolic
extract of Averrhoa carambola leaf for 5 weeks……...............................
120
Figure 4.22 HDL-C level of normal rats after treatment with 1000 mg/kg methanolic
extract of Averrhoa carambola leaf for 5 weeks………………...……....
120
Figure 4.23 VLDL-C level of normal rats after treatment with 1000 mg/kg
methanolic extract of Averrhoa carambola leaf for 5 weeks…………….
121
Figure 4.24 AI level of normal rats after treatment with 1000 mg/kg of methanolic
extract of Averrhoa carambola leaf for 5 weeks….…………………...…
121
Figure 4.25
Figure 4.26
Effect of various doses of methanolic extract of Averrhoa carambola
leaf on total cholesterol level of high fat diet-induced chronic
hyperlipidaemic rats after 5 weeks treatment…………….………………
Percentage changes of total cholesterol level of high fat diet-induced
chronic hyperlipidaemic rats after treatment with various doses of
methanolic extract of Averrhoa carambola leaf for 5 weeks…...………..
124
125
Figure 4.27 Effect of various doses of methanolic extract of Averrhoa carambola
leaf on triglycerides level of high fat diet-induced chronic
hyperlipidaemic rats after 5 weeks treatment ……...…………………….
126
Figure 4.28 Percentage changes of triglycerides level of high fat diet-induced
chronic hyperlipidaemic rats after treatment with various doses of
methanolic extract of Averrhoa carambola leaf for 5 weeks…………….
127
Figure 4.29 LDL-C level of high fat diet-induced chronic hyperlipidaemic rats after
treatment with various doses of methanolic extract of Averrhoa
xxiii
carambola leaf for 5 weeks……..……………………………………….. 128
Figure 4.30 HDL-C level of high fat diet-induced chronic hyperlipidaemic rats after
treatment with various doses of methanolic extract of Averrhoa
carambola leaf for 5 weeks…..…………………………………….........
129
Figure 4.31 VLDL-C level of high fat diet-induced chronic hyperlipidaemic rats
after treatment with various doses of methanolic extract of Averrhoa
carambola leaf for 5 weeks………………………………………………
129
Figure 4.32 AI level of high fat diet-induced chronic hyperlipidaemic rats after
treatment with various doses of methanolic extract of Averrhoa
carambola leaf for 5 weeks…..…………………………………………..
130
Figure 4.33 Average body weight of normal rats after treatment with 1000 mg/kg
methanolic extract of Averrhoa carambola leaf at day 1 and at day
45………………………………………………………….....…………...
131
Figure 4.34 Percentage changes in body weight level of normal rats after treatment
with 1000 mg/kg of methanolic extract of Averrhoa carambola leaf for
5 weeks.......................................................................................................
132
Figure 4.35 BMI values of normal rats after treatment with 1000 mg/kg of
methanolic extract of Averrhoa carambola leaf for 5
weeks………………………………..………………………...………….
132
Figure 4.36 Average daily consumed food of normal rats after treatment with 1000
mg/kg of methanolic extract of Averrhoa carambola leaf for 5
weeks…………………………………………….…………….…………
133
xxiv
Figure 4.37 Faecal dry weight values of normal rats after treatment with 1000
mg/kg methanolic extract of Averrhoa carambola leaf for 5
weeks……………………………………………..…………..…………
133
Figure 4.38 Relative liver weight of normal rats after treatment with 1000 mg/kg
methanolic extract of Averrhoa carambola leaf………..….…...………..
134
Figure 4.39 Average body weight of high fat diet-induced chronic hyperlipidaemic
rats after treatment with various doses of methanolic extract of
Averrhoa carambola leaf…………………………………………...……
137
Figure 4.40 Percentage changes in body weight levels of high fat diet-induced
chronic hyperlipidaemic rats after treatment with various doses of
methanolic extract of Averrhoa carambola leaf for 5 weeks.……………
137
Figure 4.41 BMI values of high fat diet-induced chronic hyperlipidaemic rats after
treatment with various doses of methanolic extract of Averrhoa
carambola leaf……….………………………………………...………...
138
Figure 4.42 Average daily consumed food in high fat diet-induced chronic
hyperlipidaemic rats after treatment with various doses of methanolic
extract of Averrhoa carambola leaf….……………..…………………....
139
Figure 4.43 Faecal dry weight values of high fat diet-induced chronic
hyperlipidaemic rats after treatment with various doses of methanolic
extract of Averrhoa carambola leaf……….……..…………………...….
140
Figure 4.44 Relative liver weight of high fat diet-induced chronic hyperlipidaemic
rats after treatment with various doses of methanolic extract of
xxv
Averrhoa carambola leaf…………………………………...………...…. 141
Figure
4.45a
Effects of 1000 mg/kg of methanolic extract of Averrhoa carambola
leaf on rat liver gross and histology of normal rats as assessed by H &
E staining………...…………………………………..…………………...
144
Figure
4.45b
Effects of different doses of methanolic extract of Averrhoa carambola
leaf on rat liver gross and histology of high fat diet induced-chronic
hyperlipidaemic rats as assessed by H&E staining……...……………….
145
Figure 4.46 Effect of different fractions of methanolic extract of Averrhoa
carambola leaf on total cholesterol level in p-407 induced acute
hyperlipidaemic rats……………………………………….…..…………
147
Figure 4.47 Percentage changes of total cholesterol level of different fractions of
methanolic extract of Averrhoa carambola leaf of p-407 induced acute
hyperlipidaemic rats……………………………………..……………..
148
Figure 4.48 Effect of different fractions of methanolic extract of Averrhoa
carambola leaf on triglycerides level in p-407 induced acute
hyperlipidaemic rats………………………………………......................
149
Figure 4.49 Percentage changes of triglycerides level of different fractions of
methanolic extract of Averrhoa carambola leaf of p-407 induced acute
hyperlipidaemic rats………………….……………………..……………
150
Figure 4.50 LDL-C level of p-407 induced acute hyperlipidaemic rats after
treatment with different fractions of methanolic extract of Averrhoa
carambola leaf……………..……………………..………………….…..
151
xxvi
Figure 4.51 HDL-C level of p-407 induced acute hyperlipidaemic rats after
treatment with different fractions of methanolic extract of Averrhoa
carambola leaf…………………..……………………………………….
152
Figure 4.52 VLDL-C level of p-407 induced acute hyperlipidaemic rats after
treatment with different fractions of methanolic extract of Averrhoa
carambola leaf…..…..……………………………………………….......
152
Figure 4.53 AI level of p-407 induced acute hyperlipidaemic rats after treatment
with different fractions of methanolic extract of Averrhoa carambola
leaf………………………………………………………………….…….
153
Figure 4.54 HMG-CoA reductase inhibitory activity of methanolic extract of
Averrhoa carambola leaf…..…………………….…………………...….
160
Figure 4.55 Pancreatic lipase inhibitory activity of methanol extract of Averrhoa
carambola leaf…………………...…………………….………………...
161
Figure 4.56 HMG-CoA reductase inhibitory activity of ethyl acetate fraction of
methanolic extract of Averrhoa carambola leaf…………………............
162
Figure 4.57 Pancreatic lipase inhibitory activity of ethyl acetate fraction of
methanolic extract of Averrhoa carambola leaf……...…………..……...
163
Figure 4.58 Level of liver total cholesterol of normal rats treated with 1000 mg/kg
methanolic extract of Averrhoa carambola leaf………….……………...
169
Figure 4.59 Level of liver triglycerides of normal rats treated with 1000 mg/kg of
methanolic extract of A. carambola leaf………………….…..………….
170
Figure 4.60 Effect of various doses of methanolic extract of Averrhoa carambola leaf
xxvii
on liver total cholesterol level of high fat diet-induced chronic
hyperlipidaemic rats………………………………………………...……..
171
Figure 4.61 Effect of various doses of methanolic extract of Averrhoa carambola
leaf on liver triglycerides level of high fat diet-induced chronic
hyperlipidaemic rats………..………………...…………………….…….
172
Figure 4.62 Effect of 1000 mg/kg of methanolic extract of Averrhoa carambola leaf
on faecal total cholesterol level of normal rats…………....………….….
174
Figure 4.63
Figure 4.64
Effect of 1000 mg/kg methanolic extract of Averrhoa carambola leaf on
faecal total cholesterol level of normal rats………….….……………….
Effect of 1000 mg/kg methanolic extract of Averrhoa carambola leaf on
percentage changes of faecal total cholesterol level of normal
rats…………..……………………………………………………………
174
175
Figure 4.65 Effect of 1000 mg/kg Averrhoa carambola leaf methanolic extract on
faecal bile acids level of normal rats…………..………...……………….
175
Figure 4.66 Effect of 1000 mg/kg Averrhoa carambola leaf methanolic extract on
faecal bile acids level of normal rats…..……….……….….……………...
176
Figure 4.67 Effect of 1000 mg/kg methanolic extract of Averrhoa carambola leaf on
percentage changes of fecal bile acids level of normal rats....….…....……
176
Figure 4.68 Effect of various doses of methanolic extract of Averrhoa carambola leaf
on faecal total cholesterol level of high fat diet-induced chronic
hyperlipidaemic rats………………………………………...………..……
178
Figure 4.69 Effect of various doses of methanolic extract of Averrhoa carambola
xxviii
leaf on faecal total cholesterol level of high fat diet-induced chronic
hyperlipidaemic rats………………………………………...……………
179
Figure 4.70 Effect of various doses of methanolic extract of Averrhoa carambola leaf
on percentage changes of faecal cholesterol level of high fat diet-induced
chronic hyperlipidaemic rats……………………………….…..………….
179
Figure 4.71 Effect of various doses of methanolic extract of Averrhoa carambola leaf
on faecal bile acids of high fat diet-induced chronic hyperlipidaemic
rats………………………………………………………………………....
181
Figure 4.72 Effect of various doses of methanolic extract of Averrhoa carambola leaf
on faecal bile acids levels of high fat diet-induced chronic
hyperlipidaemic rats…………………………………..…………………...
182
Figure 4.73 Effect of various doses of methanolic extract of Averrhoa carambola leaf
on percentage changes of faecal bile acids levels of high fat diet-induced
chronic hyperlipidaemic rats……………………………...……………….
182
Figure 4.74 The effect of oral administration of single dose of 5000 mg/kg
methanolic extract of Averrhoa carambola leaf on body weight of female
rats………………………………………………….……………………..
185
Figure 4.75 Body weight of female rats treated with different doses of methanolic
extract of A. carambola leaf for 28 days………….……………………….
187
Figure 4.76 Body weight of male rats treated with different doses of methanolic
extract of Averrhoa carambola leaf for 28 days…...…..……………….....
187
Figure 4.77 Effects of different doses of methanolic extract of Averrhoa carambola
xxix
leaf on liver histology of female rats in sub-chronic toxicity study for 28
days as assessed by H&E staining…..………………….………..……...
196
Figure 4.78 Effects of different doses of methanolic extract of Averrhoa carambola
leaf on kidney histology of female rats in sub-chronic toxicity study for
28 days as assessed by H&E staining..…………………………..……......
197
Figure 4.79 Effects of different doses of methanolic extract of Averrhoa carambola
leaf on liver histology of male rats in sub-chronic toxicity study for 28
days as assessed by H&E staining………………………………………...
199
Figure 4.80 Effects of different doses of methanolic extract of Averrhoa carambola
leaf on kidney histology of male rats in sub-chronic toxicity study for 28
days as assessed by H&E staining……..……………….………................
199
Figure 4.81 HPLC chromatogram for apigenin………..………………………….….... 201
Figure 4.82 UV-vis spectrum of apigenin…………………...…………………....…… 202
Figure 4.83 Calibration curve of apigenin…………………………………...……….... 202
Figure 4.84 HPLC chromatogram for methanolic extract of Averrhoa carambola
leaf……………………………………………………….………………...
205
Figure 4.85 HPLC chromatogram for ethyl acetate fraction…….….…..……………... 206
xxx
LIST OF SYMBOLS
α Alpha
γ Gamma
β Beta
< Less than
> More than
µ
n
Micro
Nano
°C
g
Celsius
Gram
xxxi
LIST OF ABBREVIATIONS
4MUO
ABC
ABTS
ACAT
AI
ARASC
BA
BMI
BSA
BW
CA
CAT
CDCA
CETP
CHD
CMC
CRI
CVD
CYP
CYP7A1
CYP27A1
CV
4-methyl umbelliferoneo
ATP-binding cassette
2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)
Acyl-coenzyme A: cholesterol acyltransferase
Atherogenic index
Animal Research and Service Centre
Bile acids
Body mass index
Bovine serum albumin
Body weight
Cholic acid
Catalase
Chenodeoxy cholic acid
Cholesteryl ester transfer protein
Coronary heart disease
Carboxymethylcellulose
Coronary risk index
Cardiovascular disease
Cytochrome P450
Cytochrom P450 for cholesterol 7α-hydroxylase
Cytochrom P450 sterol 27-hydroxylase
Coefficient of variation
xxxii
DAD
DMSO
DPPH
DTNB
FAs
FFAs
FBS
G6PD
GAE
GSH
GSH-Px
GSSG
HAT
HDL-C
HFD
HMG-CoA
HPLC
HUVEC
LCAT
LDL-C
LDLR
LPL
LPO
MAG
Diode array detector
Dimethyl sulfoxide
2, 2’-Diphenyl-1-picrylhydrazyl
5, 5-dithio-bis-nitrobenzoic acid
Fatty acids
Free fatty acids
Fetal bovine serum
Glucose -6 -phosphate dehydrogenase
Gallic acid equivalent
Reduced glutathione
Glutathione peroxidase
Oxidised glutathione
Hydrogen atom transfer
High- density lipoprotein cholesterol
High-fat diet
3-hydroxy-3-methylglutaryl coenzyme A reductase
High performance liquid chromatography
Human umbilical vein endothelial cells
Lecithin cholesterol acyl transferase
Low- density lipoprotein cholesterol
Low-density lipoprotein receptors
Lipoprotein lipase
Lipid peroxidation
Monoacylglycerol
xxxiii
MCAs
MDA
mg
µg/mL
µL
MTS
NPC1L1
OECD
P-407
PBS
PC
PGDH
PBMC
PL
PLs
PO
PPAR-α
PPLA2
PTFE
RPMI
RBCs
ROS
RNS
Muricholic acids
Malondialdehyde
Milligram
Microgram/millilitre
Microliter
3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy- phenyl)-2-
(4- sulfophenyl)-2H tetrazolium salt
Niemann-Pick C1-like 1 protein
Organization for Economic Cooperation and Development
Poloxamer- 407
Phosphate buffer saline
Phosphocholesterol
Phospho gluconate dehydrogenase
peripheral blood mononuclear cells
Pancreatic lipase
Phospholipids
Per oral
Peroxisome proliferator-activated receptor
Pancreatic phospholipase A2
Polytetrafluoroethylene
Roswell Park Memorial Institute
Red blood cells
Reactive oxygen species
Reactive nitrogen species
xxxiv
RSD
SDS
SEM
SET
SOD
SR-B1
STZ
TC
TBARS
TE
TFC
TG
TP
TPC
TPTZ
TWR-1339
T X-100
VLDL-C
Relative standard deviation
Sodium dodecyl sulfate
Standard error mean
Single electron transfer
Superoxide dismutase
Scavenger receptor class B1
Streptozotocin
Total cholesterol
Thiobarbituric acid reactive substances
Trolox equivalent
Total flavonoid content
Triglycerides
Total protein
Total phenolic content
2, 4, 6-Tri (2- pyridyl)-s-triazine
Triton-WR- 1339
Triton X-100
Very Low- density lipoprotein cholesterol
xxxv
AKTIVITI ANTIHIPERLIPIDEMIK DAN
ANTIOKSIDAN EKSTRAK BAHAGIAN BERBEZA
BAGI AVERRHOA CARAMBOLA DAN ELUSIDASI
MEKANISME TINDAKANNYA
ABSTRAK
Averrhoa carambola, biasanya dikenali sebagai belimbing merupakan salah satu
herba yang digunakan secara meluas dalam perubatan tradisional masyarakat
Malaysia, daun dan buahnya merupakan bahagian yang paling banyak digunakan.
Kajian ini bertujuan menyiasat kesan antihiperlipidemik, aktiviti anti-oksidan dan
toksisiti ekstrak metanol dan akueus bahagian yang berlainan daripada A. carambola
dengan tumpuan untuk elusidasi mekanisme tindakannya. Daripada semua ekstrak
yang diuji, ekstrak metanol bahagian daun A. carambola menunjukkan aktiviti
antihiperlipidemik terbaik dalam model tikus hiperlipidemik akut teraruh oleh
poloxamer-407 berbanding kawalan hiperlipidemik yang setanding dengan aktiviti
atorvastatin. Berikutan pemberian kronik sehingga lima minggu, tiada penurunan
signifikan diperhatikan dalam aras parameter lipid bagi tikus normal yang dirawat
dengan 1000 mg/kg ekstrak metanol daun. Sebaliknya, perbezaan yang signifikan
diperhatikan dalam parameter lipid tikus hiperlipidemik teraruh diet tinggi lemak
selepas dirawat dengan 500 dan 1000 mg/kg ekstrak metanol daun berbanding
kawalan normal. Hasil kajian ini mencadangkan ekstrak metanol daun tersebut
bertindak sebagai agen antihiperlipidemik dan bukan sebagai agen hipolipidemik.
Selepas proses pemeringkatan, ujian menggunakan tikus hiperlipidemik akut teraruh
oleh poloxamer-407 menunjukkan fraksi etil asetat bagi ekstrak methanol daun
A. carambola mempamerkan kesan paling poten dalam penurunan semua parameter
xxxvi
lipid kecuali meningkatkan aras HDL-C. Bagi penilaian antioksidan, ekstrak metanol
daun dan batang A. carambola menunjukkan aktiviti antioksidan paling tinggi.
Kandungan fenolik dan flavonoid total bagi ekstrak A. carambola menunjukkan
korelasi yang kuat dengan aktiviti antioksidan, tetapi tiada korelasi diperhatikan
dengan kesan antihiperlipidemianya. Ekstrak metanol daun dan fraksi etil asetatnya
menunjukkan kesan perencatan bergantungan dos ke atas enzim HMG-CoA
reduktase pada kepekatan 5 dan 10 mg/mL, manakala kesan perencatan yang lemah
dikesan pada enzim lipase pankreas in vitro. Tambahan lagi, ekstrak metanol daun
meningkatkan aras enzim antioksidan in vivo secara signifikan dan menurunkan aras
peroksidasi lipid dalam sampel serum dan homogenat hepar secara bergantungan
dos. Selain itu, ekstrak metanol daun yang diberikan kepada tikus diet tinggi lemak
pada dos 500 dan 1000 mg/kg menunjukkan keberkesanan dalam menurunkan
penghasilan kolesterol dan trigliserida di dalam hepar dan meningkatkan
perkumuhan kolesterol dan asid hempedu di dalam tinja. Penyiasatan menggunakan
empat titisan sel kanser (K-562, HL-60, kasumi-1 dan HCT-116) mendapati kesemua
ekstrak A. carambola tidak menunjukkan kesan sitotoksik. Kajian toksisiti akut dan
sub-kronik menunjukkan ekstrak tersebut adalah selamat dan tiada perubahan
signifikan diperhatikan bagi kedua-dua parameter biokimia dan hematologi dalam
tikus rawatan berbanding kumpulan kawalan. Secara keseluruhannya, kajian ini
mencadangkan ekstrak metanol daun A. carambola mempunyai kesan penurunan
lipid yang boleh dibangunkan selanjutnya sebagai agen antihiperlipidemik.
xxxvii
ANTIHYPERLIPIDAEMIC AND ANTIOXIDANT
ACTIVITIES OF EXTRACTS OF DIFFERENT PARTS
OF AVERRHOA CARAMBOLA AND ELUCIDATION OF
THEIR MECHANISMS OF ACTION
ABSTRACT
Averrhoa carambola, commonly known as star fruit is one of the widely used herbs
in the Malaysian traditional medicine, with the leaf and fruits being the most utilized
parts. This study aims to investigate the antihyperlipidaemic effect, antioxidant
activity and toxicity of methanolic and aqueous extracts of different parts of
A. carambola with focus on elucidating the underlying mechanism of action.
Of the tested extracts, the methanolic extract of A. carambola leaf showed the most
potent antihyperlipidaemic activity in poloxamer-407-induced acute hyperlipidaemic
rat model compared to the hyperlipidaemic control, which was comparable with that
of atorvastatin. Upon chronic administration up to five weeks, no significant decrease
was observed in the levels of the lipid parameters of normal rats treated with
1000 mg/kg of methanolic extract of leaf. In contrast, significant changes were
observed in lipid parameters of high-fat diet induced hyperlipidemic rats after treated
with 500 and 1000 mg/kg leaf methanolic extract as compared with the
hyperlipidaemic control. These findings thus suggest that methanolic extract of
A. carambola leaf works as an antihyperlipidaemic rather than a hypolipidaemic
agent. Following fractionation, assessment using poloxamer-407 induced acute
hyperlipidaemic rats showed that the ethyl acetate fraction of methanolic extract of
A. carambola leaf exhibits the most potent significant effect in terms of reducing all
lipid parameters except increasing high density lipoprotein cholesterol (HDL-C)
xxxviii
levels. For antioxidant evaluation, methanolic extract of A. carambola stem and leaf
showed the highest antioxidant activity. The total phenolic and flavonoid contents of
A. carambola extracts showed strong correlation with their antioxidant activities, but
no correlation was found with their antihyperlipidaemic effects. Methanolic extract
of leaf and its ethyl acetate fraction produced dose-dependent inhibitory effects on
HMG-CoA reductase at 5 and 10 mg/mL concentrations, while weak inhibitory
effect was detected on pancreatic lipase in vitro. In addition, methanolic extract of
the leaf significantly increased the in vivo antioxidant enzymes levels and decreased
the lipid peroxidation in liver homogenates and serum samples in a dose-dependent
manner. On the other hand, methanolic extract of leaf given to high fat-diet rats at the
doses of 500 and 1000 mg/kg was effective in reducing the synthesis of cholesterol
and triglycerides in the liver and increasing the excretion of cholesterol and bile acids
in faeces. An investigation using four cancer cell lines (K-562, HL-60, kasumi-1 and
HCT-116) revealed that none of A. carambola extracts had cytotoxic effects. Acute
and sub-chronic toxicity study of methanolic extract of A. carambola leaf showed
that the extract was safe and no significant changes was observed in both
biochemical and haematological parameters in treated rats compared with control
group. Overall, this study suggests that the methanolic extract of A. carambola leaf
has lipids lowering effect that could be further developed as an antihyperlipidaemic
agent.
1
CHAPTER 1
INTRODUCTION
1.1 Background
Cardiovascular diseases (CVDs) are responsible for the highest burden of disease
globally (Merriel et al., 2014). They are the leading causes of death, morbidity and
health expenses in developed and developing countries accounting around 30 % of
the annual global mortality and 10 % of worldwide health burden (Deales et al.,
2013; Nair and Wang, 2013). Despite of having several therapeutic measures, focus
has now been given for establishing effective preventive strategy for detecting and
controlling of cardiovascular risk factors (O'Donnell and Elosua, 2008; Valdés et al.,
2014).
Cardiovascular risk factors include a set of plasma lipids such as triglycerides
(TG), total cholesterol (TC), very low density lipoprotein-cholesterol (VLDL-C), low
density lipoprotein-cholesterol (LDL-C) and anti-atherogenic or high density
lipoprotein-cholesterol (HDL-C) (Alzaid et al., 2014; Nelson, 2013). Dyslipidaemia
is a highly heterogeneous class of metabolic disorders which is characterized by
abnormalities in serum levels of various lipoproteins. The abnormalities of
lipoproteins include elevation in TC, LDL-C and TG along with reduction in HDL-
C. It is a powerful risk factor for coronary heart disease (CHD) (Cahalin et al., 2013;
Pratt et al., 2014). Etiologically, dyslipidaemia relies on specific metabolic
backgrounds such as insulin resistance, thyroid dysfunction and defects in the
gastrointestinal absorption of cholesterol and lipids, as well as mutations in cell
2
surface receptors and enzymes (Yadav et al., 2014). Additionally, dyslipidaemia
could occur because of suboptimal diet, obesity, inactive life style, genetic deviations
and metabolism abnormalities (Xu et al., 2014).
An increase in plasma lipids concentrations (TC, TG, LDL-C, and VLDL-C)
or decreased in HDL-C levels beyond certain level give rise to physiological
condition known as hyperlipidaemia which is the widest form of dyslipidaemia
worldwide. It has also been reported to be the most widespread marker for
susceptibility to atherosclerotic heart disease (Chen et al., 2014). Oxidative
modification of LDL-C, protein glycation, glucose-auto-oxidation with production of
free radicals and lipid peroxidation products are the main factors responsible for
ischemic heart diseases which occurs as a result of hyperlipidaemia (Yang et al.,
2008).
High levels of plasma lipids, mainly cholesterol, are a common feature of
atherosclerosis, a condition in which arterial damage can lead to ischemic heart
disease, myocardial infarction and cerebrovascular coincidences (Prasad et al., 2012).
Hypercholesterolaemia and hypertriglyceridaemia are important risk factors, either
alone or together. It was found that they are extensively contributing in the
acceleration of the manifestation and development of coronary heart disease as well
as the progression of atherosclerosis (Cahalin et al., 2013; Merriel et al., 2014).
Accumulation of high levels of LDL-C in the extracellular sub-endothelial
space of arteries is highly atherogenic and toxic to vascular cells which may lead to
atherosclerosis, hypertension, obesity, diabetes and functional depression in some
organs (Catapano et al., 2000; Jain et al., 2010). Several studies documented that
3
there is an obvious correlation between high cholesterol level in serum and
cardiovascular disease (Bays et al., 2001). According to the American Heart
Association report in 2004, heart disease and stroke will become the leading cause of
death and disability worldwide. It is estimated that, by 2030, more than 24 million
per year will suffer from the cardiovascular problems (Reinhardt, 2005). Globally,
each year approximately 12 million people die due to cardiovascular diseases.
Factors such as diet high in saturated fats and cholesterol, age, family history,
hypertension and life style are of great significance but high level of cholesterol,
particularly LDL-C is mainly responsible for the occurrence of CHD (Farias et al.,
1996).
1.2 Therapeutic challenges
Hyperlipidaemia has risen to the top in terms of causes of death in both developed
and developing countries (Sunil et al., 2012). In Malaysia, the prevalence rate of
hypercholesterolaemia accounts about 35.1 % (6.2 million) of adults (18 years and
above) in which 8.4 % are known to have hypercholesterolaemia and 26.6 % are
previously undiagnosed with hypercholesterolaemia (NHMS, 2011). There are
various classes of synthetic lipid lowering agents used in current therapy belonging
to the statins, fibrates or bile acid sequestrants groups. Although, they possess
beneficial therapeutic effects, they are often associated with some serious side effects
such as rhabdomyolysis, myopathy, elevation of hepatic enzyme levels and an
increasing risk of gallstones (Javed et al., 2006; Laurance and Bennett, 1992; Shin et
al., 2014).Thus, there is an exigent need for new lipid lowering agents with high
therapeutic value and minimum tolerable side effects (Sefi et al., 2010; Shin et al.,
2014).
4
1.3 Problem statements
Previously, a study among local plants indicated that different insoluble fibers
prepared from Averrhoa carambola fruits have potential antihypocholesterolaemic
activity (Wu et al., 2009). In addition, another study investigated the in vivo effect of
micronized insoluble fiber and fiber-rich fraction from star fruit on lipids metabolism
in a murine model (Herman-Lara et al., 2014).
However, to date there is neither detailed investigation on the lipid lowering
effects of A. carambola nor report on the antihyperlipidaemic effect of other parts of
A. carambola. This has created an interest to work on various parts of A. carambola
to evaluate their antihyperlipidaemic effects and to further investigate the mechanism
of action and toxicity.
1.4 Objectives
The objectives of the present study are:
i. to evaluate the antihyperlipidaemic effects of methanolic and aqueous extracts of
different parts of A. carambola and the fractions of the most active extract in
chemically-induced acute hyperlipidaemic rats model
ii. to evaluate the antihyperlipidaemic effect of the most active extract of
A. carambola in diet-induced chronic hyperlipidaemic rats model
iii. to evaluate the antioxidant activity of methanolic and aqueous extract of different
parts of A. carambola and the fractions of most active extract
iv. to elucidate the mechanism of antihyperlidaemic effect of the most active extract
of A. carambola and its bioactive fraction on
a. inhibition of enzyme involved in lipids synthesis
5
b. lipids and bile acids absorption and excretion
c. in vivo antioxidant and lipid peroxidation
v. to investigate the toxicity of the most active extract of A. carambola
vi. to standardize the most active extract of A. carambola using selected marker
compound
The research scheme is presented in figure 1.1.
6
1.5 Flow chart of the study
Figure 1.1: Flow chart of the study
Total protein
Lipid peroxidation
Superoxide dismutase
Catalase
Reduced glutathione
Glutathione peroxidase
TPC
TFC
FRAP
ABTS
DPPH
Dose response study
Bioactivity- guided
fractionation of the most active
extract of A. carambola leaf methanolic extract
P-407 induced acute
hyperlipidaemic rats for fractions Effects on lipids &
bile acids excretions
Effect on lipid
absorption
Effect on HMG-
CoA reductase
& pancreatic
lipase enzymes
Cytotoxicity study
Antioxidant studies Toxicological
evaluation Phytochemical
analysis Antihyperlipidaemic studies
Different parts of A. carambola
Mechanism of
antihyperlipidaemic
for the most active
extract and its
bioactive fraction
In vitro antioxidant
for extracts
& fractions
In vivo antioxidant
for the most
active
extract
Standardization using
HPLC
P-407 induced
acute
hyperlipidaemic
rats model for
all extracts
High fat diet –
induced chronic
hyperlipidaemic
rats model for the
most active
extract
Sub-chronic
toxicity study
Acute
toxicity
study
Methanolic & aqueous extracts
7
CHAPTER 2
LITERATURE REVIEW
2.1 Lipids
The term ‘‘Lipid’’ is imitative from ‘‘lipos’’, which refers to animal fat or vegetable
oil. Adiposity is derived from ‘‘adipo’’ that denotes to body fat (Driskell, 2009).
Utmost amounts of body lipids are stored in the adipocytes and adipose tissue
including triglycerides and free cholesterol (Bays et al., 2013). The term lipids also
refer to an entire class of fats and fat-like substances in the blood. The most essential
lipids in the body include; fatty acids (FA), cholesterol, cholesterol esters, TGs and
phospholipids (PLs).
2.1.1 Fatty acids
Fats are defined mainly as carboxylic acids (esters) with long hydrocarbon chains
which are either saturated or unsaturated. Mostly, they are derived
from triglycerides or phospholipids. They are named "free" fatty acids because of not
attached to the other molecules. They represent an important source of energy
because they yield large quantities of ATP when metabolized (Ibrahim et al., 2013).
OH
O
1 Free fatty acid
http://en.wikipedia.org/wiki/Triglyceridehttp://en.wikipedia.org/wiki/Phospholipid
8
2.1.2 Phospholipids
Phospholipids (PLs) resemble the TG with small different in which one fatty acid in
TG is replaced by phosphate and a nitrogenous base (Ibrahim et al., 2013).
O
OO
O
O
P
O
-O
ON+
2 Phospholipids
2.1.3 Triglycerides
Triglycerides (TG) are esters consisting of a glycerol molecule attached to three fatty
acid residues. It could be found in dietary fats and can be synthesized in the liver and
adipose tissue (Phan and Tso, 2001). It offers a source of stored energy when it is
required, especially in case of starvation. It is found in all plasma lipoproteins and
are the major component of lipoproteins with density less than 1.019 kg/L (Rosenson
et al., 2002). The ideal or normal value of TG is less than 150 mg/dL (1.69 mmol/L)
and values between 150 to 199 mg/dL is considered at the borderline high, while
a values from 200 to 499 mg/dL are high and above that considered very high
(Ducharme and Radhamma, 2008, Raza et al., 2004). They are atherogenic because
they are rich in apo C-III, which delays the lipolysis of VLDL and inhibits its uptake
and clearance from plasma (Poirier et al., 2006).
H
CH
CH O
CH
H
O
O
O
O
O
3 Triglycerides
9
2.1.4 Cholesterol and cholesterol esters
Cholesterol and cholesterol esters are important elements found in all human cell
membranes. Cholesterol is an essential constituent of steroid hormones and bile
acids. They could be synthesized in liver and many tissues as well as may be
acquired from dietary fat. Their main functions are to build, repair cells and produce
hormones such as oestrogen and testosterone (Rudel et al., 2005). In addition, they
modulate cell membrane fluidity and work as a precursor of bile acids, which play an
important role in the digestion of fats (Ahmed et al., 2009).
Cholesterol molecule is an amphipathic lipid, due to the presence of
hydrophilic group (3β-hydroxyl group) attached to the hydrophobic part of the
molecule. In addition to polarity, the 3β-hydroxyl reduces cholesterol ability to form
esters (Pikuleva and Curcio, 2014). The desired value of TC is less than 200 mg/dL
(5.17 mmol/L) and value between 200 to 239 mg/dL (5.17-6.18 mmol/L) is
considered at the borderline high, while a value of 240 mg/dL(6.21 mmol/L) or more
is high (Ducharme and Radhamma, 2008).
Cholesterol is stored in the cells in the form of cholesteryl esters (one
cholesterol molecule bound to one fatty acid by an ester bond). Esterification is
carried out by Acyl-CoA: cholesterol acyltransferase (ACAT) 1 and 2. ACAT 1 is
universally expressed, while ACAT 2 is expressed only in enterocytes and
hepatocytes. Esterification of cholesterol will produce a different shape molecule,
which is greater in size and hydrophobicity (Lemaire-Ewing et al., 2012; Rudel et al.,
2005).
10
OH
CH3
H3C
CH3
H3C
CH3
H H
H
4 Cholesterol
OCR
O
H3C H3C
CH3
H H
H
CH3
CH3
5 Cholesterol ester
Free cholesterol could be eliminated from the liver into the bile via the ATP-
binding cassette (ABC) G5/G8 heterodimer. The cholesterol ring structure formed is
highly stable and not easily metabolized (Parini et al., 2004). Cholesterol and other
types of fats cannot dissolve in the blood. Thus, they have to be transported by
attachment to specific molecules called lipoproteins in order to form macromolecular
complexes (Abrass, 2004).
2.2 Lipoproteins
Lipoproteins are macromolecule complexes, which consist of spherical particles
containing a hundreds of lipids and protein molecules. The main functions of
lipoproteins is carrying and transporting the plasma lipids (Kanakavalli et al., 2014).
There are five major lipoproteins; each one has its own function: chylomicrons,
VLDLs, intermediate-density lipoproteins (IDLs), LDL-C and HDL-C (Kanakavalli
11
et al., 2014, Von Zychlinski et al., 2014). Table 2.1 demonstrates the physical
properties of lipoproteins and their contents of apolipoproteins (Babin and Gibbons,
2009; Crook, 2012; Von Zychlinski et al., 2014). Apolipoproteins are known as
protein components of the lipoproteins or apoproteins. They assist as cofactors for
enzymes and ligands for receptors. Disturbances in lipid handling will occur if there
is any defect happened in apolipoprotein metabolism (Ducharme and Radhamma,
2008).
12
Table 2.1: Physical properties of plasma lipoproteins
(Babin and Gibbons, 2009; Crook, 2012; Ducharme and Radhamma, 2008; Von
Zychlinski et al., 2014)
2.3 Bile acids
Bile acids (BA) are functional compounds that simplify emulsification, absorption,
and transportation of fats and sterols in the liver and intestine through formation of
soluble mixed micelles with lipids (Ye et al., 2013). Daily, in the liver of the adult
human about 500 mg of cholesterol is transformed into BA (Staels et al., 2010).
Chylomicron VLDL-C IDL-C LDL-C HDL-C
Source Gut Liver VLDL-C VLDL-C
via ILD-C
Gut /liver
Density g/mL
13
They are the cornerstones, which play vital role in maintenance of mammalian
cholesterol homeostasis. The liver represents the unique source and site of BA
formations (Cherrington et al., 2013).
Besides their role in lipid digestion, bile acids also denature dietary proteins,
enhancing their rate of cleavage by pancreatic proteolytic enzymes. Bile acids also
possess antimicrobial activity which remain poorly understood (Liu et al., 2013b).
Synthesis of bile acids requires a group of enzymes belonging to the cytochrome
P450 (CYP450) superfamily (Alrefai and Gill, 2007). BA biosynthesis involves
modification of the ring structure of cholesterol, oxidation and shortening of the side
chain and lastly conjugation with an ami